Mouse anti lamin a c

The following primary antibodies were used: rabbit anti-Pan-cadherin (C3678, Sigma-Aldrich, St. Louis, MO) that recognizes the conserved C-terminal domain of classic cadherins, mouse anti-N-cadherin (clone 32) and anti-E-cadherin (clone 36), both from BD Biosciences (Franklin Lakes, New Jersey, USA), rabbit anti-β-catenin (Invitrogen-Molecular Probes, Carlsbad, CA), mouse anti-active-β-catenin (clone 8E7, Millipore, Billerica, MA, USA), mouse anti-lamin A∖C (BD Biosciences), and mouse anti-α-tubulin (clone DM1a, Sigma-Aldrich). Secondary antibodies were Alexa Fluor™ 488 goat anti-rabbit IgG, Alexa Fluor™ 546 rabbit anti-mouse IgG (Invitrogen, Life Technologies, Brazil, São Paulo, SP, Brazil), and peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse (Promega, Madison, WI). DAPI dihydrochloride (Invitrogen) was used for nuclear staining. The γ-secretase activity inhibitor Dapt (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-phenylglycine-t-butyl-ester) was from Merck Biosciences (Darmstadt, Germany). Nuclear and cytoplasmic fractions were extracted using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL).

Protein extraction was performed based on our previous report [14 (link)]. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. Signals were detected using ECL detection reagent (Millipore Corporation, Burlington, MA, USA) following the manufacturer’s instructions. The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, San Jose, CA, USA), mouse anti-c-myc, mouse anti-cyclin D1, rabbit anti-HDAC1, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, Irvine, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (GeneTex) were used as appropriate. Detailed information is given in the Supplementary Materials.

Western blot assays were conducted as described previously [7 (link)]. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% or 10% gels (Thermo) and were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). PVDF membranes were blocked in 1 mg/mL BSA (Sigma-Aldrich) at room temperature for 1 h. Primary antibodies were diluted 1:2000 and incubated overnight at 4 °C. Secondary antibodies diluted 1:5000 were added and incubated at room temperature for 1 h. The band intensities corresponding to the protein samples were measured using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The primary antibodies used were as follows: mouse anti-β-catenin, mouse anti-lamin A/C (BD Biosciences, CA, USA), mouse anti-TCF4 clone 6H5-3 (Millipore), mouse anti-N-EBP1 (C11) (Santa Cruz, CA, USA), rabbit anti-EBP1, rabbit anti-histone H3, rabbit anti-α-tubulin, rabbit anti-HIF-1α and rabbit anti-β-actin (GeneTex, CA, USA). Horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) were used as appropriate.